NOTIFICACIÓN INDIVIDUAL DE LOS DESPIDOS

4.3.1 Cells and viruses. Madin-Darby canine kidney (MDCK) cells and new born

porcine tracheal epithelial (NPTr) cells were maintained in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) (Life Technologies). 293T cells were

maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS. Influenza A/Puerto Rico/8/34 (H1N1) (PR8-WT) and A/Sw/SK/18789/02 (H1N1) (SIV/SK-WT) viruses were propagated in 11-day-old embryonated chicken eggs as described previously (487). PR8 virus lacking NS1 protein (del NS1) was kindly provided by Dr. Garcia- Sastre and was propagated in Vero cells maintained in MEM with 10% FBS. PR8-WT and SIV/SK-WT virus were titrated by plaque assay on MDCK cells, while del NS1 was titrated on Vero cells. PR8 virus carrying the mutations R38A and K41A in NS1 was rescued by reverse genetics (214). The virus was propagated using a MDCK cell line stably expressing the NS1 protein (MDCK-NS1) and titrated by plaque assay on the MDCK-NS1 cell line.

4.3.2 Antibodies and reagents. Rabbit polyclonal NS1 and NP antibodies were

generated in our laboratory as previously described (488). The other antibodies were purchased from different sources as follows: Mouse anti-HA antibody and Mouse anti-Flag M2 antibody (Sigma-Aldrich), Rabbit anti-flag DYKDDDDK tag antibody (Cell signaling technology), Mouse anti-Influenza A NP AA5H antibody (AbD Serotec), Rabbit polyclonal antibody to DDX3 (Abcam), Rabbit polyclonal antibody to PABP1 (Abcam), Rabbit polyclonal to HA tag – ChIP grade (Abcam), Mouse monoclonal antibody to β-actin (Cell Signaling Technology), Goat anti-TIA-1 antibody (Santa Cruz), Donkey anti-Rabbit IgG secondary antibody, Alexa Fluor 405 (Abcam), Donkey anti-Mouse IgG secondary antibody, Alexa Fluor 488 ( Life Technologies), Donkey anti-Goat secondary antibody, Alexa Fluor 633 (Life Technologies), IRDye 680RD anti-

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Rabbit antibody (LI-COR), IRDye 800CW anti-mouse antibody (LI-COR). Horse serum used in immunofluorescent staining was purchased from Life Technologies. Stellaris FISH probes specific to PR8 M vRNA for Fluorescence in situ hybridization (FISH) assay were purchased from Biosearch technologies.

4.3.3 Transfection and immunoprecipitation (IP). To examine DDX3 interaction with

the viral proteins, 293T cells were seeded at a density of 1×106 cells/well in six-well plates. One μg of each plasmid expressing the protein of interest was transfected using TransIT-LT1 as per the manufacturer’s recommendation. For transfection in NPTr cells, Lipofectamine LTX with PLUS reagent (Life Technologies) was used as per the manufacturer’s recommendation. The plasmids used for transfection include, pcDNA-HA-DDX3, pcDNA-SK-NP, pcDNA-PR8-NP, pcDNA-PR8-NS1 and pCMV-3×Flag-PR8-NS1, pCMV-3×Flag-DDX3 (DDX3), pCMV- 3×Flag-core helicase DDX3 (DDX3-CH), pCMV-3×Flag-C-term deletion DDX3 (DDX3-del CTD) and pCMV-3×Flag-N-term deletion DDX3 (DDX3-del NTD). The protein of interest was cloned into pCMV-3×Flag and pcDNA-HA plasmids such that the Flag- or HA-tag is fused to the N-terminal sequence of the expressed fusion protein. For examining protein interactions by IP, the cell lysate was collected in 1 ml Flag lysis buffer (FLB) (50 mM Tris HCl, pH 7.4, with 150 mM NaCl, 1 mM EDTA, and 1% TRITON X-100). The cell lysate was then sonicated and cleared of cellular debris by centrifugation. For RNase A treatment, RNase A (Life

Technologies) was added to the lysate at a concentration of 10 μg/ml and incubated on ice for 30 mins before proceeding with the immunoprecipitation assay.

For IP, 1.5 μg of mouse anti-HA (Sigma-Aldrich) or mouse monoclonal anti-Flag M2 (Sigma-Aldrich) antibody or mouse anti-Influenza A NP (AbD Serotec) antibody was added to the cell lysate and incubated with gentle rocking at 4°C for 1 hr and 15 mins. Then, 35 µl of

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Dynabeads Protein G (Life Technologies) was added to the lysate and incubated for another 1 hr and 15 mins with gentle rocking at 4°C. The beads were then washed extensively with FLB and the precipitated proteins were subjected to Western blotting with appropriate antibodies. The IP data presented are representative of multiple experiments.

4.3.4 Western blotting. Samples were resolved by sodium dodecyl sulfate-10%

polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Bio-Rad). Membranes were blocked with 10% skim milk for 30 minutes and then incubated with a primary antibody diluted in Tris Buffered Sodium chloride solution with 0.1% Tween-20 (TBST) at 4°C overnight. The following primary antibodies were used for Western blotting: Rabbit polyclonal to NS1 and Rabbit polyclonal to NP (in-house generated), Rabbit anti-Flag DYKDDDDK tag antibody (Cell signaling technology), Rabbit polyclonal antibody to DDX3 (Abcam), Rabbit polyclonal to HA tag–ChIP grade (Abcam), Mouse monoclonal antibody to β- actin (Cell Signaling Technology). After washing in TBST, the membranes were incubated in TBST containing IRDye 680RD Goat anti-Rabbit IgG antibody or IRDye 800CW Goat anti- Mouse IgG antibody. Membranes were washed again in TBST and scanned using an Odyssey imager (Li-Cor Biosciences).

4.3.5 Immunofluorescent staining. NPTr cells were grown on glass chamber slides.

After the experimental treatment and/or infection, the cells were washed with PBS, fixed with 4% Paraformaldehyde (PFA) in PBS for 15 mins at room temperature and then permeabilized with ice-cold methanol for 15 mins at room temperature. The cells were washed in PBS, blocked in 5% horse serum in PBS for 45 mins and then incubated with the primary antibody in the blocking buffer for 2 hrs at room temperature or overnight at 4ºC (266). The following primary antibodies were used for immunostaining: Mouse anti-Flag M2 antibody (Sigma), Rabbit anti-

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Flag DYKDDDDK tag antibody (Cell signaling technology), Mouse anti-Influenza A NP AA5H antibody (AbD Serotec), Rabbit polyclonal antibody to DDX3 (Abcam), Rabbit polyclonal antibody to PABP1 (Abcam), Goat anti-TIA-1 antibody (Santa Cruz). The cells were again washed in PBS and incubated with the Alexa Fluor conjugated secondary antibodies in the blocking buffer for 1 hr at room temperature. After washing in PBS, the cells were mounted using Prolong Gold Antifade reagent (Life Technologies). Multiple images of different fields of view were captured using Leica TCS SP8 confocal laser microscope. A representative of the multiple images is presented in the results.

For the quantification of the number of SG forming cells, approximately 50 cells showing positive immunostaining for NP were considered from two random microscopy panels. The number of cells showing punctuate TIA-1 staining among the NP positive cells were counted visually.

4.3.6 Knockdown of DDX3. NPTr cells were plated at a density of 4×104 cells/well in 24-well plate, 3.8 ×104 cells/well in 4-well chamber slide and 8 ×104 cells/well in 12-well plate. Next day, medium was replaced with OptiMEM and the siRNA containing transfection mix was made with OptiMEM and Lipofectamine 2000 as per manufacturer’s protocol. The siRNA mixture containing four independent siRNAs targeting DDX3 (GS1654) and the Negative siRNA (SI03650318) were obtained from Qiagen. The transfection mix was added to the cells in OptiMEM for 5-6 hrs. Then, it was replaced with complete media and incubated for 48 hrs before proceeding with the experimental treatment.

4.3.7 FISH Assay. For the FISH assay, cells were washed with PBS and fixed with 4%

Paraformaldehyde (PFA) for 10 min at room temperature after virus infection. After washing with PBS, the cells were permeabilized with 70% ethanol overnight at 4°C. The cells were then

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incubated in a hybridization solution [10% dextran sulfate, 2mM vanadyl-ribonucleoside

complex, 0.02% RNA-free BSA, 1mg/ml E.coli tRNA, 2× saline-sodium citrate (SSC) and 10% formamide] containing 125 nM vRNA probe (Stellaris FISH probes, Biosearch Technologies) at 28°C overnight in the dark. The cells were then washed again and imaged with a Leica TCS SP8 confocal laser microscope after adding the mounting solution. The Stellaris FISH probes against the M vRNA segment were conjugated with the Quasar 670 Dye. The probes were custom designed using the Stellaris probe designer (version 4.1) with the sequence of the M segment as the target. A total of 39 probes were generated and this mixture of vRNA probes were used in the FISH assay to visualize the M vRNA in the infected cells.

To visualize DDX3 and NP, primary antibodies against the two proteins were added to the hybridization solution along with the vRNA probe during the overnight incubation period described above. Next day, the cells were washed and incubated with the respective Alexa Fluor secondary antibodies in the hybridization solution for 1 hr in the dark at room temperature. Cells were washed again and observed with the confocal microscope. Multiple images of different fields of view were captured using Leica TCS SP8 confocal laser microscope. A representative of the multiple images is presented in the results.

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